Lactococcus lactis strains for producing bioactive peptides having anti-hypertensive and cholesterol-lowering effects

ABSTRACT

New  Lactococcus lactis  strains, NRRL B-50571 and NRRL B-50572, and a bacterial preparation containing the same, have the ability to produce bioactive peptides that reduce blood pressure, lower LDL-cholesterol (bad cholesterol) and present antioxidant properties for better cardiovascular health. These biologically active peptides may be produced within the food for the production of a food product, such as a functional food, or they may be produced from protein sources and subsequently added to a food as part of the formulation or as part of a food supplement or a pharmaceutical preparation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/509,925, filed Oct. 8, 2014; which is a divisional application ofU.S. application Ser. No. 13/629,398, filed Sep. 27, 2012, now U.S. Pat.No. 8,865,155; which claims the benefit of U.S. Provisional ApplicationNo. 61/540,979, filed Sep. 29, 2011. The contents of the aboveapplications are incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence_Listing.txt with a creation date of Feb. 16, 2016, anda size of 4.0 kilobytes. The Sequence_Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

In general, the invention relates to Lactococcus lactis strains for theproduction of bioactive peptides. More particularly, the inventionrelates to Lactococcus lactis strains, and bacterial preparationsthereof, for the production of bioactive peptides havingantihypertensive and cholesterol-lowering effects in mammals and relatednutritional and therapeutic products.

BACKGROUND

Coronary heart disease (CHD), which is considered the most common andserious form of cardiovascular disease, is the first cause of death indeveloped industrialized countries. Hypertension and elevated bloodcholesterol levels, particularly high low density-density lipoproteincholesterol (LDL-C), are two of the major modified risk factors for thedevelopment of CHD (Department of Health and Human Services, 2000).

The long-term regulation of blood pressure is associated with therennin-angiotensin system. The conversion of angiotensin I intoangiotensin II, a potent vasoconstrictor octapeptide, by theangiotensin-converting enzyme (ACE) [EC 3.4.15.1] has long been known.Hence, the inhibition of this enzyme can reduce high arterial bloodpressure through ACE-inhibitory (ACEI) compounds. However, several sideeffects have been associated with the ACE-inhibitory drugs. On the otherhand, ACEI peptides derived from foods sources such as milk proteins areconsidered safer and without the side effects associated with the drugs.

Milk proteins have received increased attention as potential ingredientsin health-promoting functional foods. It is accepted that proteins frommilk may act as precursors of biologically active peptides withdifferent physiological effects on the digestive, endocrine,cardiovascular, immune and nervous systems (Korhonen, 2009, J. Funct.Foods 1: 177-187). Indeed, it has been reported that an effective way toincrease the amount of bioactive peptides in dairy products is by milkfermentation with highly proteolytic strains of lactic acid bacteria(LAB) (López-Fandiño et al., 2006, Int. Dairy J. 16: 1277-1293). LABgrowth in milk is dependent on the specific proteolytic systems for thegeneration of free peptides as a source of nitrogen (Hugenholtz, 2008,Int. Dairy J. 18, 466-475). Indeed, several ACEI peptides and/or withantihypertensive activity derived from milk proteins by the action ofLactobacillus helveticus and Saccharomyces cerevisae (Nakamura et al.,1995, J. Dairy Sci. 78:777-783; Nakamura et al., 1995, J. Dairy Sci.78:1253-1257) or Lactobacillus helveticus (Sipola et al, 2002, J. DairyRes. 69: 103-111; Seppo et al., 2003, Am. J. Clin. Nutr. 77:326-330)have been found. As a result, there are some commercial products, suchas Calpis sour milk drink (Calpis Co., Japan) and Evolus (Valio,Finland). Calpis sour milk is claimed as suitable for those with mildhypertension and is fermented with Lactobacillus helveticus andSaccharomyces cervisiae and Evolus which is claimed as the firstEuropean functional food to help lower blood pressure, also fermentedwith Lactobacillus helveticus. Both fermented milk products containbioactive peptides responsible for the ACE-inhibition and presentedantihypertensive effects in hypertensive rats.

These biological effects of Lactobacillus helveticus strains have beendescribed in the prior art. For instance, international patentapplication WO99/16862, Yamamoto et al., describes the strainLactobacillus helveticus CM4, FERM BP-6060 which is capable of producinga large amount of the tripeptide Val-Pro-Pro and/or Ile-Pro-Pro.Furthermore, U.S. Pat. No. 5,449,661, Nakamura et al., describes thepreparation of a peptide containing the tripeptide sequence Val-Pro-Proand its use for lowering hypertension, obtained from fermenting milkwith the strain Lactobacillus helveticus JCM 1004.

Similarly, it has been shown that peptides released by Enterococcusfaecalis strains from milk proteins were able to decrease arterial bloodpressure in spontaneously hypertensive rats (SHR) (Muguerza et al.,2006, Int. Dairy J., 16:61-69; Quirós et al., 2007, Int. Dairy J., 17,33-41). In fact, international patent application WO 2004/104182,relates to Enterococcus faecalis bacteria which can produce bioactivepeptides, such as peptides with ACE inhibitory activity and/orantihypertensive activity. Even though LAB and the specific speciesLactobacillus and Enterococccus, have been widely studied andrecommended for use for the production of health—promoting peptides,there is still constant pursuit of finding new bacteria which are usefulfor the production of bioactive peptides from dairy proteins. To thebest of our knowledge, the beneficial health effects of peptides infermented milk with Lactococcus lactis strains have not been reported.L. lactis is one of the most important LAB, since it generally takespart of commercial starter cultures used in the manufacture of fermenteddairy foods (Odamaki et al., 2011, Systematic Appl Microbiol 34,429-434). Lactococcus lactis strains are able to improve theorganoleptic characteristics of dairy products since they areresponsible for the formation of aromatic compounds (Ayad 2009, FoodMicrobiol 26, 533-541). Previous studies in our laboratory showed thatspecific L. lactis strains isolated from native ecosystems were able toproduce remarkable aroma profiles in fermented milk (Gutierrez-Méndez etal., 2008, J. Dairy Sci. 91, 49-579). Furthermore, it was reported thata wild L. lactis strain presented ACEI peptides in Mexican Fresco cheese(Torres-Llanez, et al., 2011, J. Dairy Sci., 94: 3794-3800). Also,specific wild L. lactis strains were explored for their ability toproduce ACEI activity in fermented milk (Rodríguez-Figueroa et al.,2010, J. Dairy Sci., 93: 5032-5038; Otte et al., 2011, Int. Dairy J.,21: 229-238). However, fermented milk products produced by L. lactisstrains were not tested in vivo to show any health benefits.

Therefore, there is still a great demand for finding new effectivemicrobes which are useful both as starters in fermented dairy foods andfor the production of bioactive peptides with unique health benefits.

SUMMARY

Specific Lactococcus lactis strains NRRL B-50571 and NRRL B-50572 havethe capacity to produce bioactive peptides that have remarkable capacityto generate an antihypertensive effect in mammals. Such hypotensivepeptides do not change arterial blood pressure in subjects withnormotensive arterial blood pressure; only hypertensive subjectsexperiment a reduction in arterial blood pressure. The bioactivepeptides are a viable option to reduce arterial blood pressure withoutthe secondary effects commonly produced by synthetic drugs.

Additionally, such bioactive peptides improve cardiovascular health bylowering bad (LDL) cholesterol and present antioxidant properties.Therefore, the mentioned lactic acid bacteria and the bioactive peptidesincluded in this invention may be used in pharmaceutical preparations aswell as in food products, such as functional foods.

One or more embodiments include the generation of bioactive peptides bythe action of novel Lactococcus lactis NRRL B-50571 or NRRL B-50572 on asubstrate comprising one or more proteins or its fragments, whichcontain specific amino acid sequences. These peptides could be used inan edible product such as a food product, food supplement or as apharmaceutical composition.

One or more embodiments involve the manufacture of food products withbioactive peptides as a consequence of the action of specificLactococcus lactis NRRL B-50571 and/or NRRL B-50572; or bacterialpreparation of the bioactive peptides, with or without othermicroorganisms, on a substrate within the food. Also, these bioactivepeptides may be separately produced and added to the food product, foodsupplement or pharmaceutical preparation as part of the formulation,with the purpose of reducing blood pressure, lowering LDL-cholesterol(bad cholesterol) and reducing oxidation, for better cardiovascularhealth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are diagrams showing typical RP-HPLC peptide profilescorresponding to water soluble extracts (WSE)<3 kDa fractions obtainedfrom the fermentation of milk by specific wild L. lactis strains (NRRLB-50571 and NRRL B-50572) at 1A) 214 nm, 1B) 280 nm. C=unfermented milk;

FIG. 2 presents IC₅₀ values of the peptide fractions obtained byhydrolysis of milk proteins with specific wild L. lactis strainsobtained by RP-HPLC. Data represent mean values±SD (n=3). Statisticaldifferences were considered with P<0.01, using one way ANOVA andTukey-Kramer test. F=Peptide fraction. F1-F5=obtained at 214 nm;F6=obtained at 280 nm;

FIGS. 3A and 3B are typical mass spectra corresponding to a peptidesequence collected from the WSE F1 obtained from milk fermented by L.lactis NRRL B-50571: 3A) Double-charged ion 362.9 m/z.; 3B) MS/MSSpectrum for the specified ion in A). After interpretation andcomparison in database, the fragment amino acid sequence matched withα-La (f63-68);

FIGS. 4A-4C are diagrams showing the change in blood pressure and HRduring 24 h in SHR treated with milk fermented by specific L. lactisstrains: (4A) systolic blood pressure (SBP), (4B) diastolic bloodpressure (DBP) and (4C) heart rate (HR). Positive control

captopril, negative control

A saline, whey fraction of milk fermented by L. lactis NRRL B-50572-3(35 mg protein/kg BW)

, whey fraction of milk fermented by L. lactis NRRL B-50571-3 (35 mgprotein/kg BW)

, whey fraction of milk fermented by L. lactis NRRL B-50572-5 (50 mgprotein/kg BW)

, whey fraction of milk fermented by L. lactis NRRL B-50571-5 (50 mgprotein/kg BW)

. Data is shown by means with their standard error. Each SHR group hadseven animals;

FIG. 5 is a diagram showing the change in systolic blood pressure during4 weeks of SHR treated by L. lactis fermented milk. L. lactis NRRLB-50571 fermented milk

; L. lactis NRRL B-50572 fermented milk

; captopril=Positive control

; Purified water=Negative control

. Data is shown by mean values±SEM (n=8). FM=Fermented milk;

FIG. 6 is a diagram showing the diastolic blood pressure during 4 weeksof SHR treated by L. lactis fermented milk. L. lactis NRRL B-50571fermented milk

; L. lactis NRRL B-50572 fermented milk

; captopril=Positive control

; Purified water=Negative control

. Data is shown by mean values±SEM (n=8). FM=Fermented milk;

FIG. 7 is a diagram showing plasma low-density lipoprotein cholesterolin SHR treated by L. lactis fermented milk for 4 weeks.Captopril=Positive control; Purified water=Negative control. Data isshown by mean values±SEM (n=8);

FIG. 8 is a diagram showing plasma high-density lipoprotein cholesterolin SHR treated by L. lactis fermented milk for 4 weeks.Captopril=Positive control; Purified water=Negative control. Data isshown by mean values±SEM (n=8);

FIG. 9 is a diagram showing plasma triglycerides in SHR treated by L.lactis fermented milk for 4 weeks. Captopril=Positive control; Purifiedwater=Negative control. Data is shown by mean values±SEM (n=8); and

FIG. 10 is a diagram showing plasma total cholesterol in SHR treated byL. lactis fermented milk for 4 weeks. Captopril=Positive control;Purified water=Negative control. Data is shown by mean values±SEM (n=8);

DETAILED DESCRIPTION

The following abbreviations are used throughout the present application:

-   -   L. lactis—Lactococcus lactis;    -   ACE—angiotensin I-converting enzyme;    -   WSE—water soluble extract;    -   RP-HPLC—reverse phase high performance liquid chromatography;    -   MS—mass spectrometry;    -   SHR—spontaneously hypertensive rats;    -   BW—body weight;    -   SBP—systolic blood pressure;    -   DBP—diastolic blood pressure;    -   HR—heart rate;    -   PP—pulse pressure;    -   PWV—pulse wave velocity;    -   LAB—Lactic acid bacteria;    -   cfu—colony-forming units;    -   LSD—Least significant difference;    -   SEM—mean standard error;    -   NRRL B-50571-3—milk fermented by L. lactis NRRL B-50571 (35 mg        protein/kg body weight (BW);    -   NRRL B-50572-3—milk fermented by L. lactis NRRL B-50572 (35 mg        protein/kg BW);    -   NRRL B-50571-5—milk fermented by L. lactis NRRL B-50571 (50 mg        protein/kg BW);    -   NRRL B-50572-5—milk fermented by L. lactis NRRL B-50572 (50 mg        protein/kg BW);    -   ND—not detected; and    -   Vs—versus.

Specific Lactococcus lactis strains NRRL B-50571 and NRRL B-50572 havethe ability to produce certain bioactive peptides having a remarkablecapacity for generating an antihypertensive effect in mammals. Thesenovel strains of Lactococcus lactis were deposited at the NationalCenter for Agricultural Utilization Research, United States Departmentof Agriculture, United States of America, in September, 2011, which areLactococcus lactis NRRL B-50571 and NRRL B-50572. These bacteria wereisolated from raw milk products and were Gram positive, catalasenegative and coccal-shaped organisms. These bacteria were identified asLactococcus lactis by PCR amplification of the gene acmA (Buist et al.,1995, J. Bacteriol. 177:1554-1563) with the primers PALA 4 y PALA 14(Table 1). Strains showed the classical characteristics for Lactococcus,such as positive growth at 10 C and 4% NaCl, but lack of growth at 45 Cand pH 9.6. They also presented important technological characteristicssuch as high proteolytic activity (8 h to coagulate litmus milk), andthe ability to ferment citrate, glucose, lactose and salicin in media.Similarly, when these strains were inoculated in reconstituted nonfatdry milk, they presented high acidifying activity (4.0<pH<5.0 in 24 h)and high proteolytic activity (Abs 340>0.10 in 24 h) according to theOPA (o-phtaldialdehyde method) (Church et al., 1983, J. Dairy Sci.66:1219-1227).

The bacterial strains Lactococcus lactis NRRL B-50571 or NRRL B-50572were propagated in 10 mL of sterile lactose (5 g L⁻¹) M17 broth andincubated at 30° C. for 24 h. Fresh cultures were obtained by repeatingthe same procedure. Initial starter culture were prepared by allowing L.lactis strains to reach 10⁶-10⁷ colony-forming units (cfu) mL⁻¹asenumerated on M17 agar containing lactose (5 g L⁻¹).

Production of Fermented Milk Containing Bioactive Peptides

Reconstituted nonfat dry milk (10%, w/v) was sterilized at 100° C. for20 min. A loop of L. lactis single pre-culture (7-8 log cfu mL⁻¹) ofNRRL B-50571 or NRRL B-50572 was inoculated into sterilized milk. Theinoculated milk was incubated for 12 h at 30° C. Then, cultures wereadded (3% v/v) to reconstituted nonfat dry sterilized milk to get thedifferent fermented milk batches. Incubation was carried out at 30° C.and stopped at 24 to 48 h by pasteurization at 75° C. for 1 min.

Preparation of the Water-Soluble Extracts (WSE) from Fermented Milk

Fermented milk was centrifuged at 20,000×g for 10 min at 0° C. Then,supernatants were collected and ultra-filtered through 3 kDa cut-offmembranes at 9,800×g for 6 min. Permeates were collected, filteredthrough a 0.45 μm disposable hydrophilic filter and frozen at −80° C.until analysis were done.

ACE Inhibitory Activity of WSE from Milk Fermented with L. lactisStrains NRRL B-50571 or NRRL B-50572

Water soluble extracts (<3 KDa) obtained after fermenting milk with L.lactis strains NRRL B-50571 or NRRL B-50572 presented high ACEI activity(>80%) and low IC₅₀'s (<25 μg/mL). The IC₅₀ is the amount of peptidecontent required to inhibit ACE activity by 50%. The ACE inhibitoryactivity was assayed by the method of Cushman and Cheung (Cushman andCheung, 1971, Biochem. Pharmacol. 20:1637-1648). The Cushman/Cheungmethod is based on the liberation of hippuric acid fromhippuryl-L-histidyl-L-Leucine, catalyzed by ACE. The ACE inhibitingpercentage was calculated by the following equation: Inhibitingpercentage=(A-B)/(A-C)×100%, where A is the absorbance at 228 nm ofhippuric acid free of sample, B is the absorbance at 228 nm of hippuricacid with sample, and C is the absorbance at 228 nm of hippuric acidfree of ACE and sample.

Antioxidant Activity of WSE from Milk Fermented with L. lactis StrainsNRRL B-50571 or NRRL B-50572

Water soluble extracts (<3 KDa) obtained after fermenting milk with L.lactis strains NRRL B-50571 or NRRL B-50572 presented high TROLOX (F.HOFFMAN—LAROCHE, LTD, Basel, Switzerland) equivalent antioxidantcapacity (TEAC) (>1500 μM) as determined by the ABTS method (Re et al.,1999, Free Radical Bio. Med. 26(9):1231-1237). Thus, fermented milk bythese specific L. lactis strains present the additional physiologicaleffects of reducing the detrimental effects of oxidation without theneed for the use of natural antioxidants such as vitamin E or vitamin C,which are extremely fat or water soluble, so their applications arelimited and cannot be maintained stable for long periods of time. On theother hand, the safety of synthetic antioxidants such asbutylhydroxyanisol (BHA) and butylhydroxytoluene (BHT) has becomequestioned and they are oil soluble, thus not useful for their use inaqueous systems. Due to the importance in preventing oxidation inbiological systems and to improve stability of food products subject tooxidation, the discovery of WSE obtained from the fermentation of milkwith specific L. lactis strains with good antioxidant properties,provides a new alternative for new commercial functional fermented dairyfoods.

Isolation of ACEI Peptide Fractions by Reversed-Phase High-PerformanceLiquid Chromatography (RP-HPLC) and Identification by Tandem MassSpectrometry

Peptide profiles from WSE were obtained by RP-HPLC. Separation wascarried out with a Discovery-C₁₈ (250 mm×4.6 mm, 5 μm particle size, 180Å pore size) column from SUPELCO ANALYTICAL (Bellefonte, Pa., USA) witha solvent flow rate of 0.25 mL min⁻¹. Once the column was equilibratedwith solvent A (0.04% Trifluoroacetic acid (TFA) in water), 20 μL of theWSE were injected. Peptides were eluted with an increasing gradient ofsolvent B (0.03% TFA in acetonitrile) from 0% to 45% in solvent A,during 60 min. Peptide profiles monitored at 214 nm and 280 nm werecollected from five chromatographic runs and freeze-dried to besubjected to ACEI activity analysis and IC₅₀ determination (FIGS. 1 and2). FIG. 1A shows WSE peptide fraction profiles produced by specificwild L. lactis strains monitored at 214 nm absorbance. Unfermented milkwas used as a control. The area under the curve of each peptide profilewas evaluated as an indirect measure of proteolysis. Results showedsignificant differences (P<0.01) between fermented milk peptide profilesand the control. On the other hand, the peptide profiles obtained frommilk fermented with different strains of L. lactis were similar. Thefirst peak eluted after 12 min in all samples. The largest concentrationof peptides eluted between 12 and 25 min when the concentration ofacetonitrile was between 9-13.5%, which may be related to the relativelyhydrophobic nature of the eluted peptide. On the other hand, when WSEwere monitored at 280 nm, only three peaks eluted between 16 and 20 min(FIG. 1 B). These peptides may have ACEI activity since they were ofaromatic nature.

Peptide chromatographic profiles were divided into 6 fractions andcollected for further evaluation. Peptide profiles obtained at 214 nmwere divided into F1-F5 fractions (FIG. 1 A), meanwhile peptide profilesobtained at 280 nm corresponded to F6 (FIG. 1 B). Peptide fractionsF1-F6 showed remarkable IC₅₀ values ranging from 0.034±0.002 to0.61±0.19 μg mL⁻¹ (FIG. 2). Results did not show significant difference(P>0.01) between all peptide fractions IC₅₀. However, the peptidefractions IC₅₀ values obtained from milk fermented by L. lactis strainsNRRL B-50571 (0.076±0.004 and 0.034±0.002 μg mL⁻¹ for F1 and F6,respectively) and milk fermented by L. lactis NRRL B-50572 (0.041±0.003and 0.084±0.003 μg mL⁻¹ for F1 and F2, respectively) showed the lowestvalues (FIG. 2). Therefore, the results suggest that the specific wildL. lactis strains presented have remarkable ACE-Inhibitory activity.Both strains did not present a significant difference (P>0.01) in IC₅₀values and proteolysis, which are related to ACE-Inhibitory activity.

Peptide identification was performed by analyzing the differentfractions by mass spectrometry using a 1100 Series LC/MSD Trap fromAgilent equipped with an electro spray ionization source (LC-ESI-MS).The nano column was a C₁₈-300 (150 mm×0.75 μm, 3.5 μm; (AGILENTTECHNOLOGIES, INC., Palo Alto, Calif., USA). The sample injection volumewas 1 μL. Solvent A was a mixture of water-acetonitrile-formic acid(10:90:0.1, v/v/v) and solvent B contained water-acetonitrile-formicacid (97:3:0.1, v/v/v). The gradient was based on the increment ofsolvent B which was initially set at 3% for 10 min and it took 23 moremin to reach 65%. The 0.7 μL min⁻¹ flow rate was directed into the massspectrometer via an electrospray interface. Nitrogen (99.99%) was usedas the nebulizing and drying gas and operated with an estimated heliumpressure of 5×10⁻³ bar. The needle voltage was set at 4 kV. Mass spectrawere acquired over a range of 300-2500 mass/charge (m/z). The signalthreshold to perform auto MS^(n) analyses was 30,000. The precursor ionswere isolated within a range of 4.0 m/z and fragmented with a voltageramp from 0.35 to 1.1 V. Peptide sequences were obtained from massspectrometry data using the Mascot server through theUniProtKB/Swiss-prot database sequences. Table 2 presents the identifiedsequences of peptides in the six fractions collected from milk fermentedby specific L. lactis strains associated to ACEI activity. A typicalmass spectrum of the peptide sequence DDQNPH, produced by L. lactis NRRLB-50571 fermented milk is shown in FIG. 3.

Antihypertensive Effects of Single-Dose Consumption of Milk Fermented bySpecific Lactococcus lactis Strains NRRL B-50571 or NRRL B-50572

Previous work demonstrated that milk fermented by specific Lactococcus(L.) lactis strains significantly inhibited the activity of angiotensinI-converting enzyme (ACE). However, the relationship between ACEI andthe in vivo action had to be tested. Therefore, the antihypertensive andheart rate (HR) lowering effect of milk fermented by specific L. lactisin a murine model was investigated. Spontaneously hypertensive male rats(SHR) (271±14 g) were randomized into four treatment groups: oraladministration of milk fermented by L. lactis NRRL B-50571 or L. lactisNRRL B-50572 at 35 or 50 mg protein/kg of body weight (BW). Two moregroups were fed with different solutions as controls: a saline solutionwas the negative control, meanwhile captopril (40 mg/kg BW), a provenACE inhibitor was the positive control. Blood pressure and heart ratewere monitored by the tail cuff method before treatments and 2, 4, 6 and24 h post oral administration. Results demonstrated that milk fermentedby L. lactis NRRL B-50571 as well as milk fermented by L. lactis NRRLB-50572 presented an important systolic (SBP) and diastolic bloodpressure (DBP) and HR lowering effect. Thus, milk fermented by specificL. lactis strains present potential benefits in the prevention andtreatment of cardiovascular diseases associated to hypertension inhumans.

Samples of specific L. lactis fermented milk (prepared as previouslydescribed) for the single dose bioassay were obtained by centrifugationat 20,000×g for 10) min at 0° C. The supernatants were collected andlyophilized with a freeze dryer until used. The experimental protocolwas performed with forty-two male spontaneously hypertensive male rats(SHR) (4-5 weeks old, 72±7 g body weight (BW)) obtained from HARLANLABORATORIES, INC, (Indianapolis, Ind., USA). SHR were weaned for eightweeks and their systolic blood pressure monitored during this period.Rats were randomly housed in pairs per cage at 21±2° C. with 12 hlight/dark cycles, 52±6% relative humidity and with ad libitum intake ofa standard diet (TEKLAD, Harlan Laboratories, USA) and purified water.SHR (12-13 weeks old, 271±14 g BW) were divided into six groups of sevenrats (n=7): Oral administration of saline was the negative control,meanwhile captopril (proven hypotensive drug) (40 mg/kg BW) was thepositive control. Animals were weighed before oral administration inorder to prepare the corresponding amount of lyophilized whey/kg ofanimal weight. Lyophilized whey fractions of milk fermented by L. lactisNRRL B-50572 or NRRL B-50571 were dissolved in 0.8 mL of saline.Treatments were NRRL B-50572-3 (35 mg protein/kg BW), NRRL B-50572-5 (50mg protein/kg BW), NRRL B-50571-3 (35 mg protein/kg BW) and NRRLB-50571-5 (50 mg protein/kg BW).

Conscious SHR received a single dose through a canula between 8:30 and9:30 am to eliminate circadian cycles. Animals were restrained in thewarming chamber for 20 min at 32° C. to detect pulsations through thecaudal artery. Systolic blood pressure (SBP), diastolic blood pressure(DBP) as well as heart rate (HR) were monitored before administrationand 2, 4, 6 and 24 h post-administration. Measurements were taken fivetimes using the non-invasive blood pressure system includedphotoelectric sensor, amplifier, automatic inflation cuff and software(Model 229, IITC LIFE SCIENCE, Woodland Hills, Calif., USA). The animalexperimental procedures were done following the guidelines andsupervision of the CIAD (Centro de Investigación en Alimentación yDesarrollo), A.C. Committee of Ethics for scientific research.

SBP changes are shown in FIG. 4a . Results showed the maximal SBPreductions at 6 h post oral administration. SHR treated with the wheyfractions of milk fermented by L. lactis NRRL B-50572-5 and L. lactisNRRL B-50571-3 presented the more relevant decrement of SBP, 16.7±3.5 mmHg and 17.7±4.0 mm Hg, respectively, although treatments were notsignificantly different (P<0.05). The maximum decrease at 6 h wasobserved in animals treated with captopril which was significantlydifferent from the treatments (P<0.05). However, the SBP measurements 24h post administration showed that SHR treated with the whey fraction ofmilk fermented by L. lactis NRRL B-50572-5 presented 4.3 mm Hg less thanrats that were treated with captopril. These results suggest that L.lactis NRRL B-50572-5 fermented milk may have an important residualblood pressure reducing effect. Moreover, a remarkable 15.3 mm Hg SBPdecrement between SHR that received the whey fraction of milk fermentedby L. lactis NRRL B-50572-5 and SHR treated with saline was found.Hence, blood pressure measurements suggested an absence of dosagedependent relationship between the protein content of the whey fractioncorresponding to milk fermented by L. lactis NRRL B-50571 and itsability to reduce SBP, meanwhile the whey fraction of milk fermentedwith L. lactis NRRL B-50572 was dosage dependent.

FIG. 4b shows the reduction of DBP in SHR caused by the oraladministration of the whey fraction of milk fermented by specific L.lactis strains. The highest decrement of DBP was observed at 6 h postoral administration. At the same time, no significant difference wasfound (P<0.05) when SHR were treated with whey fraction of milkfermented by L. lactis NRRL B-50571 at any protein content or wheyfraction of fermented milk L. lactis NRRL B-50572-5. Whey fractions frommilk fermented by L. lactis NRRL B-50571 as well as milk fermented withL. lactis NRRL B-50572 presented an important dosage dependentantihypertensive effect through DBP measurements. Although, captoprilgenerated the maximum DBP reduction with each measurement, there was nota significant difference (P<0.05) with the hypotensive effect of thewhey fraction of milk fermented by L. lactis NRRL B-50572-5.

HR reductions at 2, 4, 6 and 24 h of treated SHR are shown in FIG. 4c .There was not a significant difference (P<0.05) in HR presented by ratsadministered with whey fractions from milk fermented with L. lactis NRRLB-50572-5 or NRRL B-50571-3 or captopril. As in SBP and DBP, the lowestHR values were found at 6 h post administration of treatments. In fact,SHR treated with the whey fraction L. lactis NRRL B-50571-3 fermentedmilk, as well as the whey fraction L. lactis NRRL B-50572-5 fermentedmilk presented the maximal HR decrement, 16.6±9.2 and 16.9±11.5 beatsmin⁻¹, respectively. Moreover, a significant (P<0.05) HR decrement (33.4beats/min) was found in SHR that received the whey fraction from L.lactis NRRL B-50572-5 fermented milk when compared with saline treatmentat the end of the 24-h post oral administration.

Antihypertensive and Hypolipidemic Effects of Long-Term Consumption ofMilk Fermented by Specific Lactococcus lactis Strains NRRL B-50571 orNRRL B-50572

It was demonstrated that the fractions of these fermented milks, showedan acute antihypertensive and heart rate (HR)-lowering effect inspontaneously hypertensive rats after receiving a single dose. Thus, theantihypertensive and hypolipidemic effects of long-term consumption offermented milk with specific L. lactis strains were also tested in SHR.

SHR were feed ad libitum with milk fermented by L. lactis NRRL B-50571,L. lactis NRRL B-50572, captopril (40 mg/kg body weight) or purifiedwater for four weeks. Results suggested that L. lactis fermented milkspresented a significant (p<0.05) blood pressure-lowering effect. Therewas not a significant difference (p>0.05) among milk fermented by L.lactis NRRL B-50571 and captopril by the second and third week oftreatment. Additionally, milk fermented by L. lactis strains modifiedSHR lipid profiles. Milk fermented by L. lactis NRRL B-50571 and B-50572was able to reduce plasma low-density lipoprotein (LDL) cholesterol by55.4±3 mg/dL and 66.3±4 mg/dL, respectively. Thus, milk fermented by L.lactis strains may be a coadjuvant in the reduction of hypertension andhyperlipidemia and may be used as a functional food for bettercardiovascular health.

Samples of specific L. lactis fermented milk (prepared as previouslydescribed) were prepared by heating at 98° C. for 10 min to inactiveproteases and L. lactis strains. Subsequently, samples were frozen at−20° C. All fermented milk samples were daily unfrozen and homogenized(for 20 minutes before use. Thirty-two male SHR were obtained fromHarlan Laboratories, Inc., (Indianapolis, Ill., USA). The rats wererandomly housed in pairs per cage at 21±2° C. with 12 h light/darkcycles, 52±6% relative humidity and with ad libitum intake of a standarddiet (TEKLAD, Harlan Laboratories, USA) during the experiment. SHR(27-28 weeks old and 355±24 g weight) were divided into four groups ofeight rats (n=8): purified water (negative control), captopril (provenhypotensive drug, positive control) (40 mg/kg body weight (BW), milkfermented by L. lactis NRRL B-50571 and milk fermented by L. lactis NRRL50572. All SHR had free access to each treatment during three weeks aspart of the protocol. Half of the animals were sacrificed at the end ofthat period. The rest of the SHR only received purified water during onemore week before being sacrificed. A research animal protocol wasfollowed according to the guidelines established by the institutional(CIAD, A.C.) Ethics Committee. The lowering blood pressure effect ofmilk fermented by specific L. lactis strains on SHR was monitoredthrough time. Animals were deposited in restrainers in the warmingchamber for 20 min at 32° C. to detect pulsations through the caudalartery. Systolic (SBP) and diastolic (DBP) blood pressures were measuredfive times on each conscious animal before treatments and every weekduring the experiment. Measurements were obtained using the tail-cuffmethod between 9 and 12 h to eliminate circadian cycles. Thenon-invasive blood pressure system used in this experiment included aphotoelectric sensor, an amplifier, an automatic inflation cuff andsoftware (Model 229, IITC Life Science Inc., Woodland Hills, Calif.,USA).

The hypolipidemic activity of milk fermented by specific L. lactisstrains were also evaluated in SHR. Blood samples were collected underanesthesia by cardiac puncture in tubes with heparin (SARSTEDT AG & CO.,Nümbrecht, Germany). Subsequently, samples were centrifuged at 2,500rpm, 4° C. for 10 min to obtain the plasma and they were frozen at −20°C. for further studies. Triglycerides (TG), total cholesterol (TC), andhigh-density lipoprotein cholesterol (HDL-C) levels in plasma weredetermined by a commercial kit (RANDOX LABORATORIES, Kearneysville, W.Va., USA), while low density lipoprotein cholesterol (LDL-C) wascalculated as the difference between TC and HDL-C according tospecifications.

Both L. lactis fermented milks were able to reduce blood pressure duringthe experiment (FIGS. 5 and 6). Results did not show significantdifference (P>0.05) between systolic blood pressure (SBP) measurementsin the first week (FIG. 5). However, by the second week, the SBPreduction in SHR that received milk fermented by L. lactis NRRL B-50571(−20.2±3.8 mm Hg) was not statistically different (P>0.05) from thosethat received captopril (−30.1±7.1 mm Hg). In fact, by the second andthird week, SHR treated with captopril or milks fermented by L. lactisNRRL B-50571 or B-50572 presented a marked lowering-effect on SBP. Bythe fourth week of treatment, milk fermented by L. lactis NRRL B-50571was able to reduce SBP by 23.3±1.8 mm Hg, meanwhile captopril reducedSBP by 28.1±1.8 mm Hg

As it is observed in FIG. 5, the SBP lowering-effect in SHR treated withmilk fermented by L. lactis NRRL B-50571 increases with time. Indeed,the maximal SBP reduction was found by the fourth week, even thoughanimals drank only water in the last week. Thus, these results suggest aresidual SBP lowering-effect after cessation of the treatment. Milkfermented containing antihypertensive peptides administered for longperiods may extend their bioactivity even after cessation of thetreatment.

SHR treated with milk fermented by L. lactis NRRL B-50571 and B-50572presented DBP lowering-effect during the experiment (FIG. 6). As in SBP,the first week, DBP measurements were not significantly different(P>0.05) between treatments. However, by the second week, milk fermentedby L. lactis NRRL B-50571 was able to reduce DBP by 24.5±6.6 mm Hg.Meanwhile, captopril reduced DBP by 38.4±8.5 mm Hg. Furthermore, by thethird experimental week, the DBP lowering-effect was not significantlydifferent (P>0.05) between SHR treated with captopril and milk fermentedby L. lactis NRRL B-50571 or B-50572. The most important DBP reduction(49.8±3.5 mm Hg) was observed by the fourth week of treatment in SHRthat received milk fermented by L. lactis NRRL B-50571.

In addition, fermented milks were able to modify SHR lipid profiles bythe third week of treatment. SHR that received milk fermented by L.lactis NRRL B-50571 or B-50572 presented 55.4±3 mg/dL and 66.2±4 mg/dLreduction of low-density lipoprotein cholesterol (LDL-C), respectively,when compared to SHR administered purified water (FIG. 7). Similarly,results showed that milk fermented by L. lactis strains reduced HDL-Csignificantly (P<0.05) in treated SHR (FIG. 8).

Plasma triglyceride (TG) content was also decreased by 34.7±3.7 mg/dL inSHR treated with L. lactis NRRL B-50572 fermented milk when compared topurified water (FIG. 9). Additionally, plasma total cholesterol (TC)content was also reduced in treated SHR, although differences were notsignificantly different. Milk fermented by L. lactis NRRL B-50572 orB-50571 was able to reduce TC by 10±3.2 mg/dL and 8.6±2.4 mg/dL,respectively (FIG. 10).

The cholesterol lowering effect may be attributed to cholesterol removalby the L. lactis strains per se, however, this remains to be determined.On the other hand, the lowering effect on LDL-C observed in this studymay also be attributed to the ingestion by SHR of dairy protein and/orpeptides produced by L. lactis, including those from whey protein.

The use of milk fermented by specific lactic acid bacteria may beconsidered as a coadjuvant for the improvement of cardiovascular health.To the best of our knowledge, this is the first in vivo study thatshowed the antihypertensive and hypolipidemic effects of long-termconsumption of fermented milk with specific L. lactis strains. Thus,dairy products fermented with L. lactis strains, NRRL B-50571 and NRRLB-50572 may be used as functional foods with potential benefits forcardiovascular health.

TABLE 1 Primers used for the identification of Lactococcuslactis strains Primer Sequence PALA 4(5′-CTTCAACAGACAAGTCC-3′), SEQ ID NO: 22 PALA 14(5′-GATAAATGATTCCAAGC-3′), SEQ ID NO: 23

TABLE 2Identification of peptides sequences obtained from milk fermented by specificwild L. lactis strains associated to ACEI activity. ExperimentalTheoretical Sample^(a) Mass Mass Sequence ID. Protein fragment SequenceNRRL B- 723.9 724.3 1 α-La (f63-68) DDQNPH 50571 1032.8 1033.5 2α-La (f82-89) LDDDLTDDI F1 698.6 698.3 3 κ-CN (f35-40) YPSYGL 1479.01479.7 4 κ-CN (f98-110) HPHPHLSFMAIPP 1035.7 1035.5 5 α-La (f55-62)YDTQAIVQ 1386.8 1387.7 6 α-La (f100-111) DDDLTDDIMCV 585.9 585.2 7κ-CN (f35-39) YPSYG F2 505.9 585.2 8 α₅₁-CN (f62-66) AESIS F3 830.1830.5 9 (3-CN (f22-28) SITRINK 1051.4 1051.5 10 α₅₁-CN (f80-88)HIQKEDVPS 904.1 904.5 11 κ-CN (f161-169) TVQVTSTAV F4 904.3 904.5 11κ-CN (f161-169) TVQVTSTAV 1038.4 1038.6 12 α₅₂-CN (f115-124) NAVPITPTLN977.1 977.6 13 β-CN (f69-77) SLPQNIPPL F5 1716.9 1717.0 14β-CN (f194-209) QEPVLGPVRGPFPIIV 1150.4 1150.7 15 β-CN (f199-209)GPVRGPFPIIV 977.2 977.6 13 β-CN (f69-77) SLPQNIPPL 1094.4 1094.6 16κ-CN (f25-33) YIPIQYVLS F6 904.4 904.5 11 κ-CN (f161-169) TVQVTSTAV1356.7 1357.7 17 κ-CN (f157-169) PEINTVQVTSTAV 591.8 592.3 18Serotransferrin (f448-453) GYLAVA NRRL B- 1371.53 1372.7 19β-CN (f129-140) DVENLHLPLPLL 50572 698.6 698.3 3 β-CN (f35-40) YPSYGL F1549.8 550.2 20 β-Lg (f60-64) ENGEC F2 904.2 904.5 11 κ-CN (f161-169)TVQVTSTAV F3 904.2 904.5 11 κ-CN (f161-169) TVQVTSTAV F5 1150.5 1150.715 β-CN (f199-209) GPVRGPFPIIV F6 922.4 922.4 21 α-La (f86-93) TDDIMCVK^(a) = Fractions collected from milk fermented by L. lactis NRRL B-50571and NRRL B-50572.

In the foregoing specification, the invention has been described withreference to specific exemplary embodiments thereof. It will, however,be evident that various modifications and changes may be made theretowithout departing from the broader spirit and scope of the invention asset forth in the appended claims. The specification and drawings are,accordingly, to be regarded in an illustrative sense rather than arestrictive sense.

1. A method of lowering low density lipoproteins (LDL) in the blood of ahypertensive subject in need thereof, comprising administering to thesubject a composition comprising a therapeutically effective amount of afermented milk or fermented milk-based product, wherein the product ismade by fermenting a milk or milk-based starting material withLactococcus lactis strain NRRL B-50571.
 2. The method of claim 1,further comprising the step of removing unhydrolyzed protein from thefermented milk or fermented milk-based product after the fermentationstep.
 3. The method of claim 1, wherein the starting material is milk.4. The method of claim 1, wherein the starting material is a milk-basedproduct.
 5. A method of lowering blood pressure in a hypertensivesubject in need thereof, comprising administering to the subject acomposition comprising a therapeutically effective amount of a fermentedmilk or fermented milk-based product, wherein the product is made byfermenting a milk or milk-based starting material with Lactococcuslactis strain NRRL B-50571.
 6. The method of claim 5, further comprisingthe step of removing unhydrolyzed protein from the fermented milk orfermented milk-based product after the fermentation step.
 7. The methodof claim 5, wherein the starting material is milk.
 8. The method ofclaim 5, wherein the starting material is a milk-based product.